4 research outputs found
Discovery of Function in the Enolase Superfamily: d‑Mannonate and d‑Gluconate Dehydratases in the d‑Mannonate Dehydratase Subgroup
The
continued increase in the size of the protein sequence databases
as a result of advances in genome sequencing technology is overwhelming
the ability to perform experimental characterization of function.
Consequently, functions are assigned to the vast majority of proteins
via automated, homology-based methods, with the result that as many
as 50% are incorrectly annotated or unannotated (Schnoes et al. PLoS Comput. Biol. 2009, 5 (12), e1000605). This manuscript describes a
study of the d-mannonate dehydratase (ManD) subgroup of the
enolase superfamily (ENS) to investigate how function diverges as
sequence diverges. Previously, one member of the subgroup had been
experimentally characterized as ManD [dehydration of d-mannonate
to 2-keto-3-deoxy-d-mannonate (equivalently, 2-keto-3-deoxy-d-gluconate)]. In this study, 42 additional members were characterized
to sample sequence–function space in the ManD subgroup. These
were found to differ in both catalytic efficiency and substrate specificity:
(1) high efficiency (<i>k</i><sub>cat</sub>/<i>K</i><sub>M</sub> = 10<sup>3</sup> to 10<sup>4</sup> M<sup>–1</sup> s<sup>–1</sup>) for dehydration of d-mannonate,
(2) low efficiency (<i>k</i><sub>cat</sub>/<i>K</i><sub>M</sub> = 10<sup>1</sup> to 10<sup>2</sup> M<sup>–1</sup> s<sup>–1</sup>) for dehydration of d-mannonate and/or D-gluconate, and 3) no-activity with either d-mannonate
or d-gluconate (or any other acid sugar tested). Thus, the
ManD subgroup is not isofunctional and includes d-gluconate
dehydratases (GlcDs) that are divergent from the GlcDs that have been
characterized in the mandelate racemase subgroup of the ENS (Lamble
et al. FEBS Lett. 2004, 576, 133–136) (Ahmed et al. Biochem. J. 2005, 390, 529–540). These
observations signal caution for functional assignment based on sequence
homology and lay the foundation for the studies of the physiological
functions of the GlcDs and the promiscuous ManDs/GlcDs